Production of β-Lactam antibiotics

Abstract

The present invention describes a process for the production of an N-α-amino-hydroxyphenylacetyl or an N-α-aminophenylacetyl β-lactam antibiotic comprising an IPNS-catalysed conversion of a precursor tripeptide hydroxyphenylglycyl-cysteinyl-valine (HpgCV) or phenylglycyl-cysteinyl-valine (PgCV), respectively, to the N-hydroxyphenylglycyl or the N-phenylglycyl β-lactam antibiotic, respectively. The tripeptide HpgCV or the tripeptide PgCV may further be prepared by contacting the amino acids hydroxyphenylglycine (Hpg) or phenylglycine (Pg), cystein (C) and valine (V) with a non-ribosomal peptide synthetase (NRPS) to effect formation of the tripeptide HpgCV or the tripeptide PgCV, the NRPS comprising a first module M1 specific for Hpg or Pg, a second module M2 specific for C and a third module M3 specific for V An IPNS is further provided having an improved activity in this conversion, as well as an NRPS catalysing the formation of the tripeptides. Also a host cell is provided capable of fermentatively producing β-lactam antibiotics with N-α-amino-hydroxyphenylacetyl or an N-α-aminophenylacetyl side chains.

1. An isolated non-ribosomal peptide synthetase (NRPS) that catalyses formation of the tripeptide hydroxyphenylglycyl-cysteinyl-valine or the tripeptide phenylglycyl-cysteinyl-valine from hydroxyphenylglycine, cysteine and valine or from phenylglycine, cysteine and valine, respectively, the NRPS comprising
 (i) a first module M1 specific for hydroxyphenylglycine or phenylglycine comprising SEQ ID NO: 2 or SEQ ID NO: 4 or, where the first module M1 is specific for hydroxyphenylglycine the first module M1 comprises a sequence additionally selected from the sixth module of a Calcium-Dependent Antibiotic (CDA) synthetase of a  Streptomyces  coelicolor or the fourth module of a  Chloroerenomycin  Synthetase of a  Amycolatopsis  orientalis or the fifth module of a  Chloroerenomycin  Synthetase of a  Amycolatopsis  orientalis, or the seventh module of a  Complestatin  Synthetase of a  Streptomyces  lavendulae, and where the first module M1 is specific for phenylglycine the first module M1 comprises a sequence additionally selected from a C-terminal module of a SnbD protein of a  Pristinamycin  Synthetase of a  Streptomyces pristinaspirali,    
 (ii) a second module M2 specific for cysteine comprises SEQ ID NO: 6 or SEQ ID NO: 8 or a second module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of a Nocardia lactamdurans or a second module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of a  Penicillium chrysogenum  or a second module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of an  Acremonium chrysogenum  or a second module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of an  Aspergillus nidulans  or a domain of the second module of  Bacillus subtilis  RB14 Iturin Synthetase Protein ItuC, or an amino acid sequence of  Penicllium chrysogenum  enabling incorporation of the amino acid L-cysteine while being coupled to the amino acid Hpq or Pq, 
 (iii) a third module M3 specific for valine having SEQ ID NO: 10 or a third module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of a Nocardia lactamdurans or a third module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of a  Penicillium chrysogenum  or a third module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of an  Acremonium chrysogenum  or a third module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase of an  Aspergillus nidulans , the third module M3 enabling incorporation of the amino acid L-valine to hydroxyphenylglycyl-Cysteine or phenylglycyl-Cysteine. 
 
     
     
       2. The peptide synthetase of  claim 1 , wherein the first module M1 specific for hydroxyphenylglycine is obtained from at least one of a Calcium-Dependent Antibiotic Synthetase, a  Chloroerenomycin  Synthetase and a  Complestatin  Synthetase. 
     
     
       3. The peptide synthetase of  claim 1 , wherein the first module M1 specific for phenylglycine is obtained from a  Pristinamycin  Synthetase. 
     
     
       4. The peptide synthetase of  claim 1 , wherein the M2 module comprises a condensation domain that is D-specific for the donor and L-specific for the acceptor ( D C L ) that is fused to an adenylation domain obtained from the second module of an δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), wherein the  D C L  domain is heterologous to the adenylation domain. 
     
     
       5. The peptide synthetase of  claim 4 , wherein the  D C L  domain of the module M2 is obtained from the enzyme that is the source of the first module M1. 
     
     
       6. The peptide synthetase of  claim 4 , wherein the  D C L  domain of the module M2 is the condensation domain of the seventh module of a Calcium-Dependent Antibiotic Synthetase. 
     
     
       7. The peptide synthetase of  claim 4 , wherein the  D C L  domain of the module M2 is the condensation domain of the second module of an Iturin Synthetase. 
     
     
       8. The peptide synthetase of  claim 1 , further comprising adenylation and thiolation domains of the M2 module and the complete M3 module are obtained from an ACVS, preferably a bacterial or fungal ACVS.